Project Summary In the effort to understand brain function in both healthy and disease states, it is important to identify the active structures of cell-to-cell signaling neuropeptides and elucidate their cellular signaling pathways. This information enables the design of therapeutic compounds to modulate these pathways for treating a variety of human health conditions. Neuropeptides can undergo a subtle post-translational modification (PTM) that isomerizes one amino acid residue from the L-stereoisomer to the D-stereoisomer. L- to D-residue isomerization alters the three-dimensional structure of the resulting D-amino acid-containing peptide (DAACP), often leading to significantly higher biological potency and stability relative to the all-L-residue analogue. A related PTM is the formation of isoaspartate from aspartate residues to form isoaspartate-containing peptides (IsoAspPs), a modification that has been implicated in a number of neurological disorders. However, both L- to D-residue isomerization and isoaspartate formation are difficult to detect because these modifications do not change a compound?s mass or chemical composition, rendering these PTMs ?invisible? to most peptide characterization approaches. The central hypothesis is that DAACPs and IsoAspPs are present as cell-to-cell signaling peptides in many animals, including mammals, but have been mischaracterized due to technological deficiencies in detecting peptide residue isomerization. There is currently an unmet requirement for methods to detect and predict the occurrence of these functionally important PTMs. This need is addressed with a DAACP and IsoAspP discovery funnel, a new technology designed to study the synthesis and signaling of peptides that undergo isomerization, with the long-term objective being to use this information to establish the neurobiological role these PTMs play in healthy organisms and in neurological disorders. Aim 1 will develop a non-targeted method to screen for DAACPs and IsoAspPs in a variety of animals and biological tissues. The method will be used to fully characterize the suite of DAACPs and IsoAspPs present in the central nervous system of the model organism Aplysia californica, as well as in central nervous, endocrine, and heart tissues of mouse. Simultaneously, Aim 2 will create a method to identify DAACPs and IsoAspPs at the level of the single cell using on-slide enzymatic digestion coupled to sensitive single cell mass spectrometry techniques. Finally, Aim 3 will fully characterize the biosynthesis and signaling of known DAACPs in Aplysia, including identifying the first L/D-isomerase enzyme that acts on cell-to-cell signaling peptides, which will allow for the identification of homologous enzymes in other animals, including mammals. Together, these efforts will define the importance of cell-to-cell signaling DAACPs and IsoAspPs and characterize their synthesis and function throughout the nervous system. The tools developed will have wide applicability to many future investigations of cell-to-cell signaling molecules.